Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater

ABSTRACT While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 mari...

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Autores principales:	Elizabeth A. Monaghan, Kelle C. Freel, Michael S. Rappé
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	HTC Pelagibacter SAR11 cryopreserved cultivation seawater Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/dd333c920d4c474abca044b08737188f
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spelling	oai:doaj.org-article:dd333c920d4c474abca044b08737188f2021-12-02T18:44:37ZIsolation of SAR11 Marine Bacteria from Cryopreserved Seawater10.1128/mSystems.00954-202379-5077https://doaj.org/article/dd333c920d4c474abca044b08737188f2020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00954-20https://doaj.org/toc/2379-5077ABSTRACT While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. Using a raw seawater sample collected from the tropical Pacific Ocean, a subsample was diluted in seawater growth medium to create 576 2-ml dilution cultures containing 5 cells each and incubated for a high-throughput culturing (HTC) experiment, while another portion was cryopreserved in 10% glycerol. After 10 months, a cryopreserved aliquot was thawed and used to create a second cultivation experiment of 480 2-ml cultures containing 5 cells each and 470 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from the raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (92%) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater environmental sample were isolated by both approaches. IMPORTANCE High-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing them in the presence of cryoprotectants, such as glycerol, has been shown to be an effective method of storing viable cells over long time periods (i.e., years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as an inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings (i) expand the potential of high-throughput cultivation experiments to include testing when immediate isolation experiments are impractical, (ii) allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and (iii) enable cultivation experiments across a wide range of other conditions that would benefit from having source inocula available over extended periods of time.Elizabeth A. MonaghanKelle C. FreelMichael S. RappéAmerican Society for MicrobiologyarticleHTCPelagibacterSAR11cryopreservedcultivationseawaterMicrobiologyQR1-502ENmSystems, Vol 5, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	HTC Pelagibacter SAR11 cryopreserved cultivation seawater Microbiology QR1-502
spellingShingle	HTC Pelagibacter SAR11 cryopreserved cultivation seawater Microbiology QR1-502 Elizabeth A. Monaghan Kelle C. Freel Michael S. Rappé Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater
description	ABSTRACT While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. Using a raw seawater sample collected from the tropical Pacific Ocean, a subsample was diluted in seawater growth medium to create 576 2-ml dilution cultures containing 5 cells each and incubated for a high-throughput culturing (HTC) experiment, while another portion was cryopreserved in 10% glycerol. After 10 months, a cryopreserved aliquot was thawed and used to create a second cultivation experiment of 480 2-ml cultures containing 5 cells each and 470 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from the raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (92%) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater environmental sample were isolated by both approaches. IMPORTANCE High-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing them in the presence of cryoprotectants, such as glycerol, has been shown to be an effective method of storing viable cells over long time periods (i.e., years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as an inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings (i) expand the potential of high-throughput cultivation experiments to include testing when immediate isolation experiments are impractical, (ii) allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and (iii) enable cultivation experiments across a wide range of other conditions that would benefit from having source inocula available over extended periods of time.
format	article
author	Elizabeth A. Monaghan Kelle C. Freel Michael S. Rappé
author_facet	Elizabeth A. Monaghan Kelle C. Freel Michael S. Rappé
author_sort	Elizabeth A. Monaghan
title	Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater
title_short	Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater
title_full	Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater
title_fullStr	Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater
title_full_unstemmed	Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater
title_sort	isolation of sar11 marine bacteria from cryopreserved seawater
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/dd333c920d4c474abca044b08737188f
work_keys_str_mv	AT elizabethamonaghan isolationofsar11marinebacteriafromcryopreservedseawater AT kellecfreel isolationofsar11marinebacteriafromcryopreservedseawater AT michaelsrappe isolationofsar11marinebacteriafromcryopreservedseawater
_version_	1718377711225798656

Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater

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